1. Field of the Invention
The present invention relates generally to the fields of molecular and immunodiagnostics. More specifically, the present invention relates to species-specific immunoreactive protein orthologs (˜200 kDa) from Ehrlichia canis and Ehrlichia chaffeensis that are useful for species-specific diagnosis of canine ehrlichiosis and human monocytotropic ehrlichiosis.
2. Description of the Related Art
Canine monocytic ehrlichiosis is a potentially fatal tick-borne disease of dogs with worldwide distribution caused primarily by the rickettsial agent, Ehrlichia canis (Huxsoll et al., 1970). E. canis is an obligately intracellular bacterium that exhibits tropism for monocytes and macrophages (Nyindo et al., 1971), and establishes persistent infections in the vertebrate host (Harrus et al., 1998). The disease is characterized by three stages: the acute stage which lasts 2 to 4 weeks; the subclinical stage, in which dogs can remain persistently infected for years, but do not exhibit clinical signs, followed by the chronic phase, where in many dogs the disease becomes progressively worse due to bone marrow hypoplasia and the prognosis less favorable (Troy et al., 1990).
Ehrlichia canis infects and causes ehrlichiosis in animals belonging to the family Canidae. Canine ehrlichiosis consists of an acute and a chronic phase. The acute phase is characterized by fever, serous nasal and ocular discharges, anorexia, depression, and loss of weight. The chronic phase is characterized by severe pancytopenia, epistaxis, hematuria, blood in feces in addition to more severe clinical signs of the acute disease. If treated early during the course of the disease, dogs respond well to doxycycline. However, chronically infected dogs do not respond well to the antibiotic. Therefore, early diagnosis is very important for treating canine ehrlichiosis.
Treating the disease in the acute phase is important for the best prognosis. Hematologic abnormalities such as leukopenia and thrombocytopenia often provide useful evidence of canine ehrlichiosis and are important factors in the initial diagnosis (Troy et al., 1990). However, diagnosis is made difficult because the clinical presentation of canine ehrlichiosis is non-specific.
Diagnosis of canine ehrlichiosis by serologic methods such as the indirect fluorescent-antibody (IFA) test has become the standard method due to its simplicity, reliability and cost effectiveness (Troy et al., 1990). However, shortcomings of the indirect fluorescent-antibody test include the inability to make a species-specific diagnosis due to antigenic cross reactivity with other closely related Ehrlichia species that infect dogs (E. chaffeensis, E. ewingii, Anaplasma phagocytophilum, and A. platys). Subjective interpretations may also result in false-negative results, or false-positives caused by cross-reactive antigens. Other diagnostic methods such as polymerase chain reaction (PCR) have been developed for specific detection of E. canis, and were reported to be more sensitive than cell culture isolation, but this method requires specialized training and expensive equipment (McBride et al., 1996). Isolation of the organism is time consuming, and only a few laboratories have been consistently successful with this method Furthermore, additional tests characterizing the isolate are required for defining a specific etiology using this method.
Serologically cross-reactive antigens shared between E. canis and E. chaffeensis have been reported. Some of the major serologically cross-reactive proteins exhibit molecular masses of 28-30-kDa (Chen et al., 1997; Rikihisa et al., 1994), and it is now known that these proteins are encoded by homologous multigene families (Ohashi et al., 1998a, b). There are 22 and 25 homologous, but nonidentical, p28 genes that have been identified and sequenced in E. chaffeensis and E. canis, respectively. Similar intraspecies and interspecies strain homology was observed between the P28 proteins of E. canis and E. chaffeensis, explaining the serologic cross reactivity of these proteins (McBride et al., 1999).
A recent report demonstrated that the rP28 protein from E. chaffeensis was an insensitive tool in diagnosing cases of human monocytotrophic ehrlichiosis (HME) (Yu et al., 1999a). The underlying reason appears to be the variability of the P28 protein among different strains of E. chaffeensis (Yu et al., 1999b). Conversely, the P28 genes identified in E. canis are conserved among geographically dispersed strains, and the E. canis rP28 has proven to be useful for diagnosis of canine ehrlichiosis (McBride et al., 1999; Ohashi 1998a). Other homologous immunoreactive proteins including the glycoproteins in E. canis (gp 140) and E. chaffeensis (gp120) have been cloned (Yu et al., 1997, 2000). Reactivity of the rgp120 of E. chaffeensis has correlated well with the indirect fluorescent-antibody for serodiagnosis of human monocytotropic ehrlichioisis, and preliminary studies with the rgp104 of E. canis suggest that it may be a sensitive and reliable immunodiagnostic antigen (Yu et al., 1999a, 2000).
The prior art is deficient in specific antigens for serologic and molecular diagnostics for E. canis and E. chaffeensis as well as methods for such use. The present invention fulfills this longstanding need and desire in the art.